Abstract
In this study, four fusion proteins were designed, in which the N-terminal cellulose-binding module as the affinity tag was immobilized on the regenerated amorphous cellulose (RAC), and the release of the C-terminal colored proteins was detected easily and rapidly after on-resin cleavage using the free tobacco etch virus protease (TEVp) variant, or the immobilized cognate protease with a binding capacity of up to 220 mg protein per gram of RAC. The enhanced stability and repetitive use of the immobilized TEVp compensated slight loss of the catalytic efficiency toward the soluble protein substrate. On-resin cleavage and purity of the released target proteins are related to the context of the fusion tag, the incorporated linker composition, and the colored protein. Owing to low cost and high binding capacity of the RAC, the TEVp immobilized on the resin is an ideal alternative for removing fusion tag. The colored proteins are easily monitored in the on-resin process of fusion proteins, and rapid separation from RAC.
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References
Guan D, Chen Z (2014) Challenges and recent advances in affinity purification of tag-free proteins. Biotechnol Lett 36:1391–1406
Waugh DS (2011) An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif 80:283–293
Fang J, Chen L, Cheng B, Fan J (2013) Engineering soluble tobacco etch virus protease accompanies the loss of stability. Protein Expr Purif 92:29–35
Puhl AC, Giacomini C, Irazoqui G, Batista-Viera F, Villarino A, Terenzi H (2009) Covalent immobilization of tobacco-etch-virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins. Biotechnol Appl Biochem 53:165–174
Miladi B, Marjou AE, Boeuf G, Bouallagui H, Dufour F, Di Martino P, Elm’selmi A (2012) Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins. J Biotechnol 158:97–103
Wang HC, Yu CC, Liang CF, Huang LD, Hwu JR, Lin CC (2014) Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation. ChemBioChem 15:829–835
Hong J, Wang Y, Ye X, Percival Zhang YH (2008) Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. J Chromatogr A 1194:150–154
Li Y (2011) Self-cleaving fusion tags for recombinant protein production. Biotechnol Lett 33:869–881
Dian C, Eshaghi S, Urbig T, McSweeney S, Heijbel A, Salbert G, Birse D (2002) Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins. J Chromatogr B Analyt Technol Biomed Life Sci 769:133–144
Kohli BM, Ostermeier C (2003) A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process. Protein Expr Purif 28:362–367
Park KW, Webster DA, Stark BC, Howard AJ, Kim KJ (2003) Fusion protein system designed to provide color to aid in the expression and purification of proteins in Escherichia coli. Plasmid 50:169–175
Finn RD, Kapelioukh I, Paine MJ (2005) Rainbow tags: a visual tag system for recombinant protein expression and purification. Biotechniques 38:387–388
Frey S, Görlich D (2014) A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J Chromatogr A 1337:95–105
Miladi B, Dridi C, El Marjou A, Boeuf G, Bouallagui H, Dufour F, Di Martino P, Elm’selmi A (2013) An improved strategy for easy process monitoring and advanced purification of recombinant proteins. Mol Biotechnol 55:227–235
Zhu K, Zhou X, Yan Y, Mo H, Xie Y, Cheng B, Fan J (2017) Cleavage of fusion proteins on the affinity resins using the TEV protease variant. Protein Expr Purif 131:27–33
Banki MR, Feng L, Wood DW (2005) Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat Methods 2:659–661
Lan D, Huang G, Shao H, Zhang L, Ma L, Chen S, Xu A (2011) An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases. Anal Biochem 415:200–202
Raux-Deery E, Leech HK, Nakrieko KA, McLean KJ, Munro AW, Heathcote P, Rigby SE, Smith AG, Warren MJ (2005) Identification and characterization of the terminal enzyme of siroheme biosynthesis from Arabidopsis thaliana: a plastid-located sirohydrochlorin ferrochelatase containing a 2FE-2S center. J Biol Chem 280:4713–4721
Harnastai IN, Gilep AA, Usanov SA (2006) The development of an efficient system for heterologous expression of cytochrome P450s in Escherichia coli using hemA gene co-expression. Protein Expr Purif 46:47–55
Kwon SJ, de Boer AL, Petri R, Schmidt-Dannert C (2003) High-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis. Appl Environ Microbiol 69:4875–4883
Harbaugh S, Kelley-Loughnane N, Davidson M, Narayanan L, Trott S, Chushak YG, Stone MO (2009) FRET-based optical assay for monitoring riboswitch activation. Biomacromol 10:1055–1160
Chen X, Pham E, Truong K (2010) TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector. Protein Sci 19:2379–2388
Kim SW, Kim JB, Lee WS, Jung WH, Ryu JM, Jang HW, Jo YB, Jung JK, Kim JH (2007) Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides. Protein Expr Purif 55:159–165
Mitchell SF, Lorsch JR (2015) Protein affinity purification using intein/chitin binding protein tags. Methods Enzymol 559:111–125
Banki MR, Gerngross TU, Wood DW (2005) Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Sci 14:1387–1395
Guillén D, Moreno-Mendieta S, Aguilera P, Sánchez S, Farres A, Rodríguez-Sanoja R (2013) The starch-binding domain as a tool for recombinant protein purification. Appl Microbiol Biotechnol 97:4141–4148
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This work was supported by Scientific and Technological Project of Anhui Province (1506c085007).
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449_2017_1772_MOESM1_ESM.tif
Figure S1. Purification of CBM-TEVpH6. The eluent from Ni–NTA agarose or proteins bound to RAC are mixed with SDS-PAGE loading buffer, boiled for 10 min, centrifuged, and analyzed. M: protein molecular weight marker. Lane 1: the eluent from Ni–NTA agarose. Lane 2: proteins bound to RAC. Arrow indicates the position of CBM-TEVpH6 (TIFF 1873 kb)
449_2017_1772_MOESM2_ESM.tif
Figure S2 Time course cleavage of GST-tevS-eDAL with the immobilized or free TEVp construct. After cleavage, the mixture is analyzed by SDS-PAGE. a Cleavage of fusion protein substrate with the immobilized TEVp construct. b Cleavage of fusion protein substrate with the free TEVp construct. M: protein molecular weight marker. The purified fusion protein and/or cleaved products are indicated by arrows. Incubation time is denoted on the top of the gel. c Activity of the released eDAL (TIFF 4423 kb)
449_2017_1772_MOESM3_ESM.tif
Figure S3 The CBM-tevS-VHb bound RAC with incubation of the inactive and active TEVp constructs. a Visual detection of the fusion protein incubated the protease constructs. b Visual detection of the RAC matrices and eluents treated with the proteases. c SDS-PAGE analysis. M: protein molecular weight marker. All of resins, eluents, and proteins on the SDS-PAGE gel, are treated correspondently as CBM-2tevS-VHb (TIFF 5335 kb)
449_2017_1772_MOESM4_ESM.tif
Figure S4 Detection of the introduced flexible linker on improving cleavage efficiency of the fusion protein GST-tevS-ZmSR bound to glutathione Sepharose 4B. a Cleavage in solution analysis. M: protein molecular weight marker. Lane 1: purified GST-tevS-ZmSR, Lane 2: the fusion protein substrate is cleaved in solution. b On-resin cleavage of the fusion protein with the tevS. Lane 1: purified GST-tevS-ZmSR. Lane 2: eluents after on-resin cleavage. Lane 3: the proteins bound to the affinity resin. c On-resin cleavage of the fusion protein with incorporation of the H6D4 linker between GST and tevS. The protein sample in each lane is corresponded with the figure S4 b (TIFF 7250 kb)
449_2017_1772_MOESM5_ESM.tif
Figure S5 Incubation of the CBM-tevS-VHb in crude extracts with the soluble CBM-TEVpH6 construct. The mass ratio of the fusion protein amount in crude extracts by determined Bradford method to the soluble TEVp construct purified by Ni–NTA resin is 5:1. The mixture is reacted at 30 °C for 3 h and analyzed by 15% SDS-PAGE. M: protein molecular weight marker. Arrows indicate the TEVp constructs, the fusion protein substrate and cleaved products. Lane 1: the fusion protein incubated with the TEVpC151A construct. Lane 2: the fusion protein incubated with the active TEVp construct (TIFF 2495 kb)
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Yu, X., Sun, J., Wang, W. et al. Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose. Bioprocess Biosyst Eng 40, 1101–1110 (2017). https://doi.org/10.1007/s00449-017-1772-4
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DOI: https://doi.org/10.1007/s00449-017-1772-4